Participation of dectin-1 receptor on NETs release against Paracoccidioides brasiliensis: role on extracellular killing

Paracoccidioides brasiliensis is a dimorphic fungus from the Paracoccidioides genus, which is the causative agent of paracoccidioidomycosis, a chronic, subacute or acute mycosis, with visceral and cutaneous involvement. This disease that is acquired through inhalation primarily attacks the lungs but, can spread to other organs. Phagocytic cells as neutrophils play an important role during innate immune response against this fungus, but studies on antifungal activities of these cells are scarce. In addition to their ability to eliminate pathogens by phagocytosis and antimicrobial secretions, neutrophils can trap and kill microorganisms by release of extracellular structures composed by DNA and antimicrobial proteins, called neutrophil extracellular traps (NETs). Here, we provide evidence that P. brasiliensis virulent strain (P. brasiliensis 18) induces NETs release. These structures were well evidenced by scanning electron microscopy, and specific NETs compounds such as histone, elastase and DNA were shown by confocal microscopy. In addition, we have shown that dectin-1 receptor is the main PRR to which fungus binds to induce NETS release. Fungi were ensnared by NETs, denoting the role of these structures in confining the fungus, avoiding dissemination. NETs were also shown to be involved in fungus killing, since fungicidal activity detected before and mainly after neutrophils activation with TNF-α, IFN-γ and GM-CSF was significantly inhibited by cocultures treatment with DNAse.

Dectin-1 is essential for reverse transcytosis of glycosylated SIgA-antigen complexes by intestinal M cells.

Intestinal microfold (M) cells possess a high transcytosis capacity and are able to transport a broad range of materials including particulate antigens, soluble macromolecules, and pathogens from the intestinal lumen to inductive sites of the mucosal immune system. M cells are also the primary pathway for delivery of secretory IgA (SIgA) to the gut-associated lymphoid tissue. However, although the consequences of SIgA uptake by M cells are now well known and described, the mechanisms whereby SIgA is selectively bound and taken up remain poorly understood. Here we first demonstrate that both the Cα1 region and glycosylation, more particularly sialic acid residues, are involved in M cell-mediated reverse transcytosis. Second, we found that SIgA is taken up by M cells via the Dectin-1 receptor, with the possible involvement of Siglec-5 acting as a co-receptor. Third, we establish that transcytosed SIgA is taken up by mucosal CX3CR1⁺ dendritic cells (DCs) via the DC-SIGN receptor. Fourth, we show that mucosal and systemic antibody responses against the HIV p24-SIgA complexes administered orally is strictly dependent on the expression of Dectin-1. Having deciphered the mechanisms leading to specific targeting of SIgA-based Ag complexes paves the way to the use of such a vehicle for mucosal vaccination against various infectious diseases.

Human neutrophils produce IL-12, IL-10, PGE2 and LTB4 in response to Paracoccidioides brasiliensis. Involvement of TLR2, mannose receptor and dectin-1

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Histoplasma capsulatum Cell Wall beta-Glucan Induces Lipid Body Formation through CD18, TLR2, and Dectin-1 Receptors: Correlation with Leukotriene B(4) Generation and Role in HIV-1 Infection

Histoplasma capsulatum (Hc) is a facultative, intracellular parasite of worldwide significance. Infection with Hc produces a broad spectrum of diseases and may progress to a life-threatening systemic disease, particularly in individuals with HIV infection. Resolution of histoplasmosis is associated with the activation of cell-mediated immunity, and leukotriene B(4) plays an important role in this event. Lipid bodies (LBs) are increasingly being recognized as multifunctional organelles with roles in inflammation and infection. In this study, we investigated LB formation in histoplasmosis and its putative function in innate immunity. LB formation in leukocytes harvested from Hc-infected C57BL/6 mice peaks on day 2 postinfection and correlates with enhanced generation of lipid mediators, including leukotriene B(4) and PGE(2). Pretreatment of leukocytes with platelet-activating factor and BLT1 receptor antagonists showed that both lipid mediators are involved in cell signaling for LB formation. Alveolar leukocytes cultured with live or dead Hc also presented an increase in LB numbers. The yeast alkali-insoluble fraction 1, which contains mainly beta-glucan isolated from the Hc cell wall, induced a dose- and time-dependent increase in LB numbers, indicating that beta-glucan plays a signaling role in LB formation. In agreement with this hypothesis, beta-glucan-elicited LB formation was inhibited in leukocytes from 5-LO(-/-), CD18(low) and TLR2(-/-) mice, as well as in leukocytes pretreated with anti-Dectin-1 Ab. Interestingly, human monocytes from HIV-1-infected patients failed to produce LBs after beta-glucan stimulation. These results demonstrate that Hc induces LB formation, an event correlated with eicosanoid production, and suggest a role for these lipid-enriched organelles in host defense during fungal infection. The Journal of Immunology, 2009, 182: 4025-4035.

Syk-Dependent Cytokine Induction by Dectin-1 Reveals a Novel Pattern Recognition Pathway for C Type Lectins

Pattern-recognition receptors (PRRs) detect molecular signatures of microbes and initiate immune responses to infection. Prototypical PRRs such as Toll-like receptors (TLRs) signal via a conserved pathway to induce innate response genes. In contrast, the signaling pathways engaged by other classes of putative PRRs remain ill defined. Here, we demonstrate that the β-glucan receptor Dectin-1, a yeast binding C type lectin known to synergize with TLR2 to induce TNFα and IL-12, can also promote synthesis of IL-2 and IL-10 through phosphorylation of the membrane proximal tyrosine in the cytoplasmic domain and recruitment of Syk kinase. syk−/− dendritic cells (DCs) do not make IL-10 or IL-2 upon yeast stimulation but produce IL-12, indicating that the Dectin-1/Syk and Dectin-1/TLR2 pathways can operate independently. These results identify a novel signaling pathway involved in pattern recognition by C type lectins and suggest a potential role for Syk kinase in regulation of innate immunity.

Autoimmune regulator (AIRE) contributes to Dectin-1-induced TNF-alpha production and complexes with caspase recruitment domain-containing protein 9 (CARD9), spleen tyrosine kinase (Syk), and Dectin-1

Background: Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome is a complex immunologic disease caused by mutation of the autoimmune regulator (AIRE) gene. Autoimmunity in patients with APECED syndrome has been shown to result from deficiency of AIRE function in transcriptional regulation of thymic peripheral tissue antigens, which leads to defective T-cell negative selection. Candidal susceptibility in patients with APECED syndrome is thought to result from aberrant adaptive immunity. Objective: To determine whether AIRE could function in anticandidal innate immune signaling, we investigated an extrathymic role for AIRE in the immune recognition of beta-glucan through the Dectin-1 pathway, which is required for defense against Candida species. Methods: Innate immune signaling through the Dectin-1 pathway was assessed in both PBMCs from patients with APECED syndrome and a monocytic cell line. Subcellular localization of AIRE was assessed by using confocal microscopy. Results: PBMCs from patients with APECED syndrome had reduced TNF-alpha responses after Dectin-1 ligation but in part used a Raf-1-mediated pathway to preserve function. In the THP-1 human monocytic cell line, reducing AIRE expression resulted in significantly decreased TNF-a release after Dectin-1 ligation. AIRE formed a transient complex with the known Dectin-1 pathway components phosphorylated spleen tyrosine kinase and caspase recruitment domain-containing protein 9 after receptor ligation and localized with Dectin-1 at the cell membrane. Conclusion: AIRE can participate in the Dectin-1 signaling pathway, indicating a novel extrathymic role for AIRE and a defect that likely contributes to fungal susceptibility in patients with APECED syndrome. (J Allergy Clin Immunol 2012;129:464-72.)

Macrophage Dectin-1 Expression Is Controlled by Leukotriene B-4 via a GM-CSF/PU.1 Axis

Pattern recognition receptors for fungi include dectin-1 and mannose receptor, and these mediate phagocytosis, as well as production of cytokines, reactive oxygen species, and the lipid mediator leukotriene B-4 (LTB4). The influence of G protein-coupled receptor ligands such as LTB4 on fungal pattern recognition receptor expression is unknown. In this study, we investigated the role of LTB4 signaling in dectin-1 expression and responsiveness in macrophages. Genetic and pharmacologic approaches showed that LTB4 production and signaling through its high-affinity G protein-coupled receptor leukotriene B4 receptor 1 (BLT1) direct dectin-1-dependent binding, ingestion, and cytokine production both in vitro and in vivo. Impaired responses to fungal glucans correlated with lower dectin-1 expression in macrophages from leukotriene (LT)- and BLT1-deficent mice than their wildtype counterparts. LTB4 increased the expression of the transcription factor responsible for dectin-1 expression, PU.1, and PU.1 small interfering RNA abolished LTB4-enhanced dectin-1 expression. GM-CSF controls PU.1 expression, and this cytokine was decreased in LT-deficient macrophages. Addition of GM-CSF to LT-deficient cells restored expression of dectin-1 and PU.1, as well as dectin-1 responsiveness. In addition, LTB4 effects on dectin-1, PU.1, and cytokine production were blunted in GM-CSF-/- macrophages. Our results identify LTB4-BLT1 signaling as an unrecognized controller of dectin-1 transcription via GM-CSF and PU.1 that is required for fungi-protective host responses. The Journal of Immunology, 2012, 189: 906-915.

The Analgesic Effect Of Dipyrone In Peripheral Tissue Involves Two Different Mechanisms: Neuronal K(atp) Channel Opening And Cb(1) Receptor Activation.

Dipyrone (metamizole) is an analgesic pro-drug used to control moderate pain. It is metabolized in two major bioactive metabolites: 4-methylaminoantipyrine (4-MAA) and 4-aminoantipyrine (4-AA). The aim of this study was to investigate the participation of peripheral CB1 and CB2 cannabinoid receptors activation in the anti-hyperalgesic effect of dipyrone, 4-MAA or 4-AA. PGE2 (100ng/50µL/paw) was locally administered in the hindpaw of male Wistar rats, and the mechanical nociceptive threshold was quantified by electronic von Frey test, before and 3h after its injection. Dipyrone, 4-MAA or 4-AA was administered 30min before the von Frey test. The selective CB1 receptor antagonist AM251, CB2 receptor antagonist AM630, cGMP inhibitor ODQ or KATP channel blocker glibenclamide were administered 30min before dipyrone, 4-MAA or 4-AA. The antisense-ODN against CB1 receptor expression was intrathecally administered once a day during four consecutive days. PGE2-induced mechanical hyperalgesia was inhibited by dipyrone, 4-MAA, and 4-AA in a dose-response manner. AM251 or ODN anti-sense against neuronal CB1 receptor, but not AM630, reversed the anti-hyperalgesic effect mediated by 4-AA, but not by dipyrone or 4-MAA. On the other hand, the anti-hyperalgesic effect of dipyrone or 4-MAA was reversed by glibenclamide or ODQ. These results suggest that the activation of neuronal CB1, but not CB2 receptor, in peripheral tissue is involved in the anti-hyperalgesic effect of 4-aminoantipyrine. In addition, 4-methylaminoantipyrine mediates the anti-hyperalgesic effect by cGMP activation and KATP opening.

Chronic ethanol intake modulates vascular levels of endothelin-1 receptor and enhances the pressor response to endothelin-1 in anaesthetized rats

Background and purpose: The contribution of endothelin-1 (ET-1) to vascular hyper-reactivity associated with chronic ethanol intake, a major risk factor in several cardiovascular diseases, remains to be investigated. Experimental approach: The biphasic haemodynamic responses to ET-1 (0.01-0.1 nmol kg(-1), i.v.) or to the selective ET(B) agonist, IRL1620 (0.001-1.0 nmol kg(-1), i.v.), with or without ET(A) or ET(B) antagonists (BQ123 (c(DTrp-Dasp-Pro-Dval-Leu)) at 1 and 2.5 mg kg(-1) and BQ788 (N-cis-2,6-dimethyl-piperidinocarbonyl-L-gamma-methylleucyl1-D-1methoxycarbonyltryptophanyl-D-norleucine) at 0.25 mg kg(-1), respectively) were tested in anaesthetized rats, after 2 weeks` chronic ethanol treatment. Hepatic parameters and ET receptor protein levels were also determined. Key results: The initial hypotensive responses to ET-1 or IRL1620 were unaffected by chronic ethanol intake, whereas the subsequent pressor effects induced by ET-1, but not by IRL1620, were potentiated. BQ123 at 2.5 but not 1 mg kg(-1) reduced the pressor responses to ET-1 in ethanol-treated rats. Conversely, BQ788 (0.25 mg kg(-1)) potentiated ET-1-induced increases in mean arterial blood pressure in control as well as in ethanol-treated rats. Interestingly, in the latter group, increases in heart rate, induced by ET-1 at a dose of 0.025 mg kg(-1) were enhanced following ET(B) receptor blockade. Finally, we observed higher levels of ET(A) receptor in the heart and mesenteric artery and a reduction of ET(B) receptor protein levels in the aorta and kidney from rats chronically treated with ethanol. Conclusions and implications: Increased vascular reactivity to ET-1 and altered protein levels of ET(A) and ET(B) receptors could play a role in the pathogenesis of cardiovascular complications associated with chronic ethanol consumption.

Ameloblast apoptosis and IGF-1 receptor expression in the continuously erupting rat incisor model

Enamel-producing cells (ameloblasts) pass through several phenotypic and functional stages during enamel formation. In the transition between secretory and maturation stages, about one quarter of the ameloblasts suddenly undergo apoptosis. We have studied this phenomenon using the continuously erupting rat incisor model. A special feature of this model is that all stages of ameloblast differentiation are presented within a single longitudinal section of the developing tooth. This permits investigation of the temporal sequence of gene and growth factor receptor expression during ameloblast differentiation and apoptosis. We describe the light and electron microscopic morphology of ameloblast apoptosis and the pattern of insulin-like growth factor-1 receptor expression by ameloblasts in the continuously erupting rat incisor model. In the developing rat incisor, ameloblast apoptosis is associated with downregulated expression of the insulin-like growth factor-1 receptor. These data are consistent with the hypothesis that ameloblasts are hard wired for apoptosis and that insulin-like growth factor-1 receptor expression is required to block the default apoptotic pathway. Possible mechanisms of insulin-like growth factor-1 inhibition of ameloblast apoptosis are presented. The rat incisor model may be useful in studies of physiological apoptosis as it presents apoptosis in a predictable pattern in adult tissues.

Maternal and neonatal interleukin-1 receptor antagonist genotype and pregnancy outcome in a population with a high rate of pre-term birth

Problem We evaluated associations between a length polymorphism in intron 2 of the gene coding for IL-1ra (gene symbol IL1RN) and pregnancy outcome in a population with a high rate of preterm birth. Method of study Subjects were pregnant women in Maceio, Brazil and their newborns. DNA was tested for IL1RN genotypes and alleles by gene amplification using primer pairs that spanned the polymorphic region. Every subject completed a detailed questionnaire. Results The frequency of allele 2 (IL1RN*2) carriage was elevated in mothers with a spontaneous preterm birth (SPTB) in the current pregnancy (P = 0.02) and also with a prior preterm delivery (P = .01). Both SPTB with intact membranes (P = 0.01) and SPTB preceded by pre-term pre-mature rupture of membranes (P = .03) were associated with IL1RN*2 carriage. A previous fetal demise was more than twice as prevalent in mothers positive for two copies of IL1RN*2. Conclusion Maternal carriage of IL1RN*2 increases susceptibility to inflammation-triggered spontaneous pre-term birth.

Regulation of CSF-1 receptor expression

Cells of the mononuclear phagocyte lineage possess receptors for macrophage colony-stimulating factor (CSF-1) encoded by the c-fms protooncogene and respond to CSF-1 with increased survival, growth, differentiation, and reversible changes in function. The c-fms gene is itself a macrophage differentiation marker. In whole mount analyses of mRNA expression in embryos, c-fms is expressed at very high levels on placental trophoblasts. It is detectable on individual cells in the yolk sac around 8.5 to 9 days postcoitus, appears on isolated cells in the head of the embryo around 9.5 dpc, and appears on numerous cells throughout the embryo by day 10.5. The extent of c-fms expression is much greater than for other macrophage-specific genes including lysozyme and a macrophage-specific protein tyrosine phosphatase. Our studies of the cis-acting elements of the c-fms promoter have indicated a key role for collaboration between the macrophage-specific transcription factor, Pu.1, which functions in determining the site of transcription initiation, and other members of the Ets transcription factor family. This is emerging as a common pattern in macrophage-specific promoters. We have shown that two PU box elements alone can function as a macrophage-specific promoter. The activity of both the artifical promoter and the c-fms promoter is activated synergistically by coexpression of Pu.1 and another Ets factor, c-Ets-2. A 3.5kb c-fms exon 2 promoter (but not the 300bp proximal promoter) is also active in a wide diversity of tumor cell lines. The interesting exception is the melanoma cell line K1735, in which the promoter is completely shut down and expression of c-fms causes growth arrest and cell death. The activity of the exon 2 promoter in these nonmacrophages is at least as serum responsive as the classic serum-responsive promoter of the c-fos gene. It is further inducible in nonmacrophages by coexpression of the c-fms product. Unlike other CSF-1/c-fms-responsive promoters, the c-fms promoter is not responsive to activated Ras even when c-Ets-2 is coexpressed. In most lines, production of full length c-fms is prevented by a downstream intronic terminator, but in Lewis lung carcinoma, read-through does occur, and expression of both c-fms and other macrophage-specific genes such as lysozyme and urokinase becomes detectable in conditions of serum deprivation. (C) 1997 Wiley-Liss, Inc.

Interleukin-1 receptor antagonist gene (IL1RN) polymorphism possibly associated to severity of rheumatic carditis in a Brazilian cohort

Aims: To evaluate the IL1RN polymorphism as a possible marker for Rheumatic Fever (RF) susceptibility or disease severity. Methods: The genotypes of 84 RF patients (Jones criteria) and 84 normal race-matched controls were determined through the analysis of the number of 86-bp tandem repeats in the second intron of IL1RN. The DNA was extracted from peripheral-blood leukocytes and amplified with specific primers. Clinical manifestations of RF were obtained through a standardized questionnaire and an extensive chart review. Carditis was defined as new onset cardiac murmur that was perceived by a trained physician with corresponding valvae regurgitation or stenosis on echocardiogram. Carditis was classified as severe in the presence of congestive heart failure or upon the indication for cardiac surgery. The statistical association among the genotypes, RF and its clinical variations was determined. Results: The presence of allele I and the genotype A1/A1 were found less frequently among patients with severe carditis when compared to patients without this manifestation (OR = 0.11, p = 0.031; OR = 0.092, p = 0.017). Neither allele I nor allele 2 were associated with the presence of RF (p = 0.188 and p = 0.106), overall carditis (p = 0.578 and p = 0.767), polyarthritis (p = 0.343 and p = 0.313) and chorea (p = 0.654 and p = 0.633). Conclusion: In the Brazilian population, the polymorphism of the IL-1ra gene is a relevant factor for rheumatic heart disease severity. (C) 2009 Elsevier Ltd. All rights reserved.

AT(1) receptor participates in the cardiac hypertrophy induced by resistance training in rats

Resistance training is accompanied by cardiac hypertrophy, but the role of the renin-angiotensin system (RAS) in this response is elusive. We evaluated this question in 36 male Wistar rats divided into six groups: control (n = 6); trained (n = 6); control + losartan (10 mg.kg(-1).day(-1), n = 6); trained + losartan (n = 6); control + high-salt diet (1%, n = 6); and trained + high-salt diet (1%, n = 6). High salt was used to inhibit the systemic RAS and losartan to block the AT(1) receptor. The exercise protocol consisted of: 4 x 12 bouts, 5x/wk during 8 wk, with 65-75% of one repetition maximum. Left ventricle weight-to-body weight ratio increased only in trained and trained + high-salt diet groups (8.5% and 10.6%, P < 0.05) compared with control. Also, none of the pathological cardiac hypertrophy markers, atrial natriuretic peptide, and alpha MHC (alpha-myosin heavy chain)-to-beta MHC ratio, were changed. ACE activity was analyzed by fluorometric assay (systemic and cardiac) and plasma renin activity (PRA) by RIA and remained unchanged upon resistance training, whereas PRA decreased significantly with the high-salt diet. Interestingly, using Western blot analysis and RT-PRC, no changes were observed in cardiac AT(2) receptor levels, whereas the AT(1) receptor gene (56%, P < 0.05) and protein (31%, P < 0.05) expressions were upregulated in the trained group. Also, cardiac ANG II concentration evaluated by ELISA remained unchanged (23.27 +/- 2.4 vs. 22.01 +/- 0.8 pg/mg, P > 0.05). Administration of a subhypotensive dose of losartan prevented left ventricle hypertrophy in response to the resistance training. Altogether, we provide evidence that resistance training-induced cardiac hypertrophy is accompanied by induction of AT(1) receptor expression with no changes in cardiac ANG II, which suggests a local activation of the RAS consistent with the hypertrophic response.

Topical interleukin-1 receptor antagonist inhibits inflammatory cell infiltration into the cornea

Interleukin (IL)-1 alpha and beta are important modulators of many functions of corneal epithelial and stromal cells that occur following injury to the cornea, including the influx of bone marrow-derived inflammatory cells into the stroma attracted by chemokines released from the stroma and epithelium. In this study, we examined the effect of topical soluble IL-1 receptor antagonist on bone marrow-derived cell influx following corneal epithelial scrape injury in a mouse model. C57BL/6 mice underwent corneal epithelial scrape followed by application of IL-1 receptor antagonist (Amgen, Thousand Oaks, CA) at a concentration of 20 mg/ml or vehicle for 24 h prior to immunocytochemical detection of marker CD11b-positive cells into the stroma. In two experiments, topical IL-1 receptor antagonist had a marked effect in blocking cell influx. For example, in experiment 1, topical IL-1 receptor antagonist markedly reduced detectible CD11b-positive cells into the corneal stroma at 24 It after epithelial injury compared with the vehicle control (3.5 +/- 0.5 (standard error of the mean) cells/400x field and 13.9 +/- 1.2 cells/400x field, respectively, p < 0.01). A second experiment with a different observer performing cell counting had the same result. Thus, the data demonstrate conclusively that topical IL-1 receptor antagonist markedly down-regulates CD-11b-positive monocytic cell appearance in the corneal stroma. Topical IL-1 receptor antagonist could be an effective adjuvant for clinical treatment of corneal conditions in which unwanted inflammation has a role in the pathophysiology of the disorder. (c) 2008 Elsevier Ltd. All rights reserved.